MOLECULAR MEDICINE REPORTS 16: 4119-4126, 2017

miR‑127 contributes to ventilator‑induced lung injury

QIAN LI1*, YA‑LI GE2*, MIN LI3, XIANG‑ZHI FANG2, YAN‑PING YUAN1,
LEI LIANG4 and SHAO‑QIANG HUANG1

1
Department of Anesthesiology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200433; 2Department of
Anesthesiology, Subei People's Hospital of Jiangsu, Yangzhou, Jiangsu 225001; 3Department of Anesthesiology,
Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000; 4Department of Colorectal Surgery,
Fudan University Shanghai Cancer Center, Shanghai 200032, P.R. China

Received June 1, 2016; Accepted April 27, 2017

DOI: 10.3892/mmr.2017.7109

Abstract. Although it is essential in critical care medicine,
mechanical ventilation often results in ventilator‑induced
lung injury (VILI). Treating mice with lipopolysaccharide
has been reported to upregulate the expression of miR‑127,
which has been implicated in the modulation of immune
responses. However, the putative roles of miR‑127 during the
development of VILI have yet to be elucidated. The present
study demonstrated that challenging mice with mechanical
ventilation for 6 h significantly upregulated the expression
of miR‑127 in bronchoalveolar lavage fluid, serum and lung
tissue samples. Conversely, following the downregulation
of miR‑127 expression in vivo using an adenovirus delivery
system, VILI‑associated pathologies, including alterations
in the pulmonary wet/dry ratio, pulmonary permeability,
lung neutrophil infiltration and levels of pro‑inflammatory
cytokines, were significantly attenuated. In addition, miR‑127
knockdown inhibited the ventilation‑induced activation of
nuclear factor (NF)‑κ B and p38 mitogen‑activated protein
kinase (MAPK). These findings suggested that the upregula-
tion of miR‑127 expression may contribute to the development
of VILI, through the modulation of pulmonary permeability,
the induction of histopathological alterations, and the poten-
tiation of inflammatory responses involving NF‑κ B and p38
MAPK‑associated signaling pathways.
Introduction
Mechanical ventilation is a life‑saving intervention for criti-
cally ill patients with acute respiratory failure. However, the
Correspondence to: Dr Shao‑Qiang Huang, Department of
Anesthesiology, Obstetrics and Gynecology Hospital, Fudan
University, 220 Handan Road, Shanghai 200433, P.R. China
E‑mail: drhuangsq@163.com
*
Contributed equally
Key words: miR‑127, lung injury, mechanical ventilation,
inflammation
ventilation procedure has been associated with the development
of ventilator‑induced lung injury (VILI) (1). The pathogenesis
of VILI has been reported to involve excessive, uncontrolled
inflammatory responses in the lungs; however, the molecular
mechanisms underlying the development of VILI have yet
to be elucidated (2,3). Inflammatory responses during the
pathogenesis of VILI have been revealed to exacerbate lung
damage, including alveolar‑capillary barrier injury, and can
result in the development of alveolar edema (4). Therefore,
the need to elucidate the molecular mechanisms implicated in
the progression of VILI is imperative for the development of
novel preventive and therapeutic approaches for the treatment
of patients with VILI.
MicroRNAs (miRNAs) are a class of small, endogenous,
noncoding RNA molecules with a length of ~22 nucleotides,
that are involved in the post‑transcriptional regulation of gene
expression, via targeting the 3' untranslated regions of target
genes (5). miR‑127 has been revealed to be highly expressed
in embryos and has been implicated in lung development,
placenta formation and cellular apoptosis (6). Aberrant
miR‑127 expression has been associated with an increased risk
of prostate, bladder and colon cancer (7,8). Previous studies
have identified miRNAs as critical regulators during lung
and systemic inflammatory responses (9,10), thus suggesting
that miRNAs may be involved in the pathogenesis of VILI,
which has a significant inflammatory component (11). Notably,
miR‑127 has been reported to be upregulated in inflam-
matory pulmonary disorders, including lung fibrosis and
bleomycin or immunocomplex‑induced lung injury (12,13).
In addition, miR‑127 is upregulated in response to lipopoly-
saccharide‑induced stimulation of macrophages (14), thus
suggesting that miR‑127 may be involved in the modulation of
immune responses.
In response to mechanical stress, a series of intracellular
signaling cascades are initiated, ultimately leading to the
excessive production of pro‑inflammatory cytokines, including
tumor necrosis factor (TNF)‑α, interleukin (IL)‑1β and IL‑6,
as well as neutrophil recruitment to the lungs (15,16). The
present study aimed to investigate whether mechanical ventila-
tion may alter the expression of miR‑127, and whether aberrant
miR‑127 expression may contribute to the development of
VILI. Therefore, a mouse model of VILI was employed to
study the involvement of miR‑127 in the pathogenesis of VILI.
4120 LI et al: miR-127 CONTRIBUTES TO LUNG INJURY
Materials and methods
Animals. A total of 128 C57BL/6 female mice (age, 6 weeks;
weight, 20‑25 g) were obtained from the Center of Comparative
Medicine of Yangzhou University (Yangzhou, China).
Animals were housed at 24±2˚C, under 12‑h light/dark cycles
with free access to food and water. The study was approved
by the Laboratory Animal Ethics Committee of Yangzhou
University Medical Academy. Mice were anesthetized prior
to tracheotomy through intraperitoneal administration of
carbrital (40 mg/kg).
Adenoviral plasmids encoding miR‑127‑specific short hairpin
(sh)RNA. Adenoviral plasmids encoding miR‑127‑specific
(UGU​GAU​CAC​UGU​CUC​CAG​CCU​GCU​GAA​GCU​CAG) and
control non‑specific (UUG​G CA​U UG​UGC​ACU​ACA​CCC​
UGC​UUU​AAU​UU) shRNAs were obtained by Thermo Fisher
Scientific, Inc. (Waltham, MA, USA). The miR‑127‑targeting
and control plasmids were termed Ad‑shmiR‑127 and
Ad‑shNeg, respectively. Adenoviruses were prepared by
Thermo Fisher Scientific, Inc. and the viral titer was deter-
mined by infecting these adenovirus cultures at a multiplicity
of infection of 50. Viral stocks with titers of 1011‑1012 infectious
units/ml were used in experiments.
Mouse model of VILI. A total of 1.3 ml Ad‑shmiR‑127 (n=18)
or Ad‑shNeg (n=18) virus was administered (as viral particles)
to each mouse via hydrodynamic injection into the tail
vein (17). On day 3 following injection, mice were tracheoto-
mized through the insertion of a blunted 20‑gauge needle into
the trachea. Mice were ventilated for 6 h using a small‑animal
ventilator (DW3000; Huaibei Zhenghua Biologic Apparatus
Facilities Co., Ltd., Huaibei, China), with a high tidal volume
of 30 ml/kg and without positive end expiratory pressure. Mice
in the control group were allowed to breathe spontaneously
for 6 h. Mice in the VILI group received only mechanical
ventilation and no adenovirus infection. Mice were sacrificed
following mechanical ventilation, and tissue samples were
harvested for further analysis.
Reverse transcription‑quantitative polymerase chain reaction
(RT‑qPCR). RT‑qPCR was used to assess the expression levels
of miR‑127 (forward, 5'‑ATG​TCA​CTG​T TA​ACC​AGC​CTG​
CT‑3' and reverse, 3'‑TTA​ACT​TAC​GAG​G GA​G GC​AGA​
TGA‑5'), as previously described (18). All reagents, primers
and probes were obtained from Invitrogen; Thermo Fisher
Scientific, Inc. Total RNA was extracted using TRIzol® reagent
and cDNA was synthesized using the PrimeScript™ RT reagent
kit (Takara Biotechnology Co., Ltd., Dalian, China), according
to the manufacturer's protocol. A TaqMan® miRNA assay
(Applied Biosystems; Thermo Fisher Scientific, Inc.) was used
to quantify the expression levels of miR‑127, according to the
manufacturer's protocol. The following thermocycling condi-
tions were used: Denaturation at 95˚C for 3 min, followed by
40 cycles of 95˚C for 12 sec and 62˚C for 40 sec, and then a
melting curve program from 62 to 95˚C (read every 0.2˚C, held
for 2 sec). U6 small nuclear RNA (forward, 5'‑AAA​GCA​AAT​
CAT​CGG​ACG​ACC‑3' and reverse, 3'‑GTA​CAA​CAC​ATT​GTT​
TCC​TCG​GA‑5') was used as the internal control. Relative gene
expression was calculated according to the 2‑ΔΔCq method (19).
Lung water content and histological evaluation. The upper
left lung was weighed, dried in an oven at 80˚C for 72 h, then
weighed again to calculate the pulmonary wet/dry (W/D) ratio.
Lung tissues were fixed at room temperature for 24 h in a 4%
paraformaldehyde‑buffered solution, paraffin‑embedded and
then cut into sections (3 µm). At room temperature, paraffin
sections were dewaxed twice in xylene for 15 and 20 min,
respectively, after drying for 45 min, then treated with a
descending ethanol series and washed in tap water for 2 min.
Following staining with hematoxylin for 5 min and washing
with tap water, the slides were differentiated in 1% acid alcohol,
blued in 1% ammonia water, counterstained with eosin for
1 min and washed with tap water again. They were then dehy-
drated by a descending ethanol series, cleared twice in xylene
for 10 min each and mounted with neutral gum. The sections
were examined and photographed by an Olympus CX21 light
microscope (magnification, x400; Olympus Corporation,
Tokyo, Japan). The degree of lung injury was assessed as
previously described by Murakami et al (20). Briefly, the
extent of edema, congestion, inflammatory cell infiltration and
hemorrhage were graded according to the following 5‑point
scale: Absent, 0; light, 1; moderate, 2; strong, 3; and intense, 4.
The scores for each histopathological feature were summed to
obtain a total score for each animal.
Pulmonary microvascular permeability. Ventilation‑induced
changes in pulmonary microvascular permeability were
assessed using a modification of the Evans blue dye extrava-
sation technique, as previously described (21). Briefly, 30 min
prior to the initiation of mechanical ventilation, 1% Evans blue
dye (Beijing Solarbio Science & Technology Co., Ltd., Beijing,
China) dissolved in PBS was injected into the tail vein of mice
at a dose of 30 mg/kg. Following ventilation, blood samples
were collected via right ventricular puncture and plasma was
isolated via centrifugation at 3,000 x g for 10 min at 4˚C. Lungs
were perfused free of blood by injecting the right ventricle with
10 ml of sterile PBS. The left lung was isolated and homog-
enized in 0.5 ml of formamide (Sigma‑Aldrich; Merck KGaA,
Darmstadt, Germany). Lung homogenates were incubated over-
night at 60˚C, then centrifuged at 4,000 x g for 30 min at 4˚C.
The optical density of the lung homogenates and diluted plasma
samples was measured at 620 and 740 nm and the permeability
index was calculated, as previously described (22).
Total cell and neutrophil counts in bronchoalveolar lavage
(BAL) fluid. Following mechanical ventilation for 6 h, BAL
fluid samples were obtained via cannulating the trachea with a
blunt 20‑gauge needle and slowly infusing the lungs with 1 ml
of ice‑cold PBS, then withdrawing the needle. The procedure
was performed three times. Total cell counts in BAL fluid
were determined using a hemocytometer. A cytocentrifuged
spin preparation (CF‑RD; Sakura Finetek Europe B.V.,
Flemingweg, Netherlands) of the BAL fluid was stained with
10% Wright‑Giemsa for 30 min at room temperature in order
to measure the percentage of neutrophils. The remainder of the
BAL fluid was centrifuged at 4,000 x g for 10 min at 4˚C and
the cell‑free supernatant was stored at ‑80˚C.
Pro‑inflammatory cytokine and total protein levels in BAL
fluid. The cell‑free BAL supernatant was thawed, and the levels
 MOLECULAR MEDICINE REPORTS 16: 4119-4126, 2017 4121

of the pro‑inflammatory cytokines TNF‑α (cat no. SMTA00B),

IL‑1β (cat no. SMLB00C) and IL‑6 (cat no. D6050) (all from
R&D Systems, Inc., Minneapolis, MN, USA) were determined
using murine cytokine‑specific Quantikine® ELISA kits,
according to the manufacturer's protocol. Total protein concen-
tration was measured using a bicinchoninic acid (BCA) protein
assay kit (cat no. P0009; Beyotime Institute of Biotechnology,
Haimen, China), according to the manufacturer's protocol.

Pulmonary neutrophil infiltration. Lung myeloperoxidase

(MPO) activity was assessed as an index of lung neutrophil
infiltration. Briefly, lung tissue (1 g) was homogenized in 1 M
PBS (pH 7.4), freeze‑thawed three times in liquid nitrogen,
and centrifuged at 12,000 x g at 4˚C for 10 min. The super-
natants were collected and MPO activity was assessed using a
commercially available kit (Nanjing Jiancheng Bioengineering
Institute, China), according to the manufacturer's protocol.

Western blot analysis. The harvested lung tissue was weighed,

homogenized in protein lysis buffer (cat no. P0013B; Beyotime
Institute of Biotechnology), and centrifuged at 12,000 x g
and 4˚C for 15 min. Total protein concentration in lung tissue
homogenates was assessed using a BCA protein assay kit (cat
no. P0009; Beyotime Institute of Biotechnology), according to
the manufacturer's protocol, using bovine serum albumin as the
calibration standard. Equal amounts (20 µl) of protein samples
were resolved by 10% SDS‑PAGE and transferred onto poly-
vinylidene difluoride membranes. The membrane was blocked
with 5% non‑fat milk in TBST buffer (10 mM Tris‑HCl, pH 7.5,
150 mM NaCl and 1.2% Tween‑20) at room temperature for
1 h. Membranes were incubated with the following primary
antibodies: Anti‑inhibitor IκB (cat no. ab32518; 1:300; Abcam,
Cambridge, UK), anti‑phosphorylated (p)‑Iκ B (cat no. 2859;
1:500), anti‑p‑p38 mitogen‑activated protein kinase (MAPK;
cat no. 4511; 1:500), anti‑p‑extracellular signal‑activated kinase
(cat no.14227; 1:500) (all from Cell Signaling Technology, Inc.,
Danvers, MA, USA) and anti‑β‑actin antibody (cat no. AA128;
1:500; Beyotime Institute of Biotechnology) at 4˚C for 12 h.
Subsequently, membranes were incubated at room temperature
for 1 h with the secondary antibody conjugated to horseradish
Figure 1. VILI upregulates miR‑127 expression. Mice in the VILI group
peroxidase (cat no.  A0208; 1:200; Beyotime Institute of
received mechanical ventilation for 6 h. Mice in the control group were allowed
Biotechnology). Protein bands were visualized using enhanced to breathe spontaneously. Reverse transcription‑quantitative polymerase
chemiluminescence with an ECL Western Blotting system chain reaction was used to assess miR‑127 expression. miR‑127 expression
(Bio‑Rad Laboratories, Inc., Hercules, CA, USA). levels were significantly upregulated in (A) BAL fluid, (B) peripheral blood
and (C) lung tissue samples isolated from the mice. Data are expressed as the
mean ± standard deviation (n=18/group). *P<0.05. VILI, ventilator‑induced
Statistical a nalysis. Data were expressed as the lung injury; BAL, bronchoalveolar lavage; miR, microRNA..
mean ± standard deviation. Statistical analysis was performed
using SPSS software version 19.0 (IBM Corp., Armonk, NY,
USA). Significance of the differences between groups was
assessed using unpaired Student's t‑test for pair‑wise compari- was significantly increased in ventilated mice compared with
sons or one‑way analysis of variance followed by a post hoc mice in the control group, which did not receive mechanical
Bonferroni test for multiple comparisons. P<0.05 was consid- ventilation (P<0.05; Fig. 1A). Similarly, miR‑127 expression
ered to indicate a statistically significant difference. was upregulated in peripheral blood and lung tissue samples
from ventilated mice, compared with mice in the control group
Results (P<0.05; Fig. 1B and C). These results suggested that miR‑127
may be implicated in the development of VILI.
VILI upregulates miR‑127 expression. To investigate the puta-
tive roles of miR‑127 in the pathogenesis of VILI, miR‑127 Silencing of miR‑127 expression following mechanical venti‑
expression was assessed in BAL fluid isolated from mice that lation via RNA interference. In order to investigate the role of
received mechanical ventilation for 6 h. miR‑127 expression miR‑127 in VILI, a mouse model with knockdown miR‑127 was
4122 LI et al: miR-127 CONTRIBUTES TO LUNG INJURY

Figure 2. Silencing of miR‑127 expression in vivo using RNA interference. (A) miR‑127 expression levels were assessed using RT‑qPCR in BAL fluid,
peripheral blood (serum) and lung tissue samples of mice infected with Ad‑shmiR‑127 adenovirus for the indicated time points. Data for the Ad‑shmiR‑127
samples are presented as fold expression relative to the Ad‑shNeg control samples. *P<0.05 vs. 1st day following injection (in BAL fluid, peripheral serum and
lung tissue samples); +P<0.05 vs. 5th day following injection (in BAL fluid, peripheral serum and lung tissue samples); &P<0.05 vs. 7th day following injection
(in BAL fluid, peripheral serum and lung tissue samples). miR‑127 expression levels were assessed using RT‑qPCR in (B) BAL fluid, (C) peripheral blood
and (D) lung tissue samples of mice infected with either Ad‑shmiR‑127 or Ad‑shNeg adenovirus for 3 days and then mechanically‑ventilated for 6 h. Data are
expressed as the mean ± standard deviation (n=18/group). *P<0.05. sh, short hairpin; BAL, bronchoalveolar lavage; RT‑qPCR, reverse transcription‑quantitative
polymerase chain reaction; VILI, ventilator‑induced lung injury; miR/miRNA, microRNA.

established via a hydrodynamic injection of Ad‑shmiR‑127 in the
tail vein. A separate group of mice were infected with adenovirus
encoding the negative‑control, non‑targeting shRNA, Ad‑shNeg.
To confirm that the recombinant adenovirus was successfully
transduced in vivo, miR‑127 expression in BAL fluid, peripheral
blood and lung tissue was assayed on the 1st, 3rd, 5th and 7th
day following the Ad‑shmiR‑127 injection. On day 3 following
viral infection, miR‑127 expression in BAL fluid, peripheral
blood and lung tissue samples was significantly decreased in
mice infected with Ad‑shmiR‑127, when compared with the 1st,
5th and 7th day following the injection (P<0.05; Fig. 2A). In
addition, miR‑127 expression was significantly downregulated
in the Ad‑shmiR‑127 group following mechanical ventilation
compared with the Ad‑shNeg group (P<0.05; Fig. 2B‑D). These
results indicated that the in vivo adenovirus‑mediated knock-
down of miR‑127 was successful in the present study, and was
thus used for further experiments to explore the role of miR‑127
in the pathogenesis of VILI.
Silencing miR‑127 expression attenuates ventilation‑induced
pulmonary histopathological alterations. Adenovirus‑mediated
miR‑127 knockdown was used to examine the effects of
miR‑127 downregulation on the development of VILI.
Following mechanical ventilation, tissue samples isolated from
mice infected with Ad‑shmiR‑127 exhibited markedly reduced
inflammatory infiltration, vascular congestion, interstitial
edema and hemorrhage compared with tissue samples from
mice infected with Ad‑shNeg (Fig. 3A). In addition, following
miR‑127 knockdown, mice demonstrated significantly lower
lung injury scores compared with non‑infected ventilated mice
(P<0.05; Fig. 3B), whereas their lung wet/dry ratio was compa-
rable to control mice (P<0.05; Fig. 3C).
Silencing miR‑127 expression attenuates ventilation‑induced
impairment of the alveolar‑capillary barrier. Using Evans
blue dye perfusion and histological analysis, the permeability
of the alveolar‑capillary barrier was assessed following
mechanical ventilation. The present results revealed that the
integrity of the alveolar‑capillary barrier was significantly
impaired in mechanically‑ventilated mice, compared with
control mice that received no ventilation (P<0.05; Fig. 3D).
Notably, following miR‑127 knockdown, alveolar‑capillary
 MOLECULAR MEDICINE REPORTS 16: 4119-4126, 2017 4123

Figure 3. Silencing of miR‑127 expression attenuates ventilation‑induced pulmonary histopathological alterations. Mice were infected with either Ad‑shmiR‑127
or Ad‑shNeg adenovirus for 3 days and then mechanically‑ventilated for 6 h. Control mice were allowed to breathe spontaneously and received no ventilation.
(A) Lung tissue samples were isolated, stained with hematoxylin and eosin, and observed under x400 magnification. The arrows indicate the following: II,
inflammatory infiltration; VC, vascular congestion; IE, interstitial edema; H, hemorrhage. miR‑127 knockdown significantly attenuated the (B) lung injury
score, (C) pulmonary wet/dry ratio, (D) alveolar‑capillary barrier permeability as indicated by Evans blue extravasation and (E) total protein concentration in
BAL fluid samples. Data are expressed as the mean ± standard deviation (n=18/group). *P<0.05, with comparisons indicated by lines. sh, short hairpin; BAL,
bronchoalveolar lavage; VILI, ventilator‑induced lung injury; miR, microRNA.

barrier permeability was significantly decreased compared
with the Ad‑shNeg group  (P<0.05;  Fig. 3D). Furthermore,
following knockdown of miR‑127 expression, total protein
levels in the BAL fluid of mechanically‑ventilated mice were
significantly decreased, thus suggesting that the integrity of
the alveolar‑capillary barrier was preserved (P<0.05; Fig. 3E).
Silencing miR‑127 expression attenuates ventilation‑induced
pulmonary inflammation. Since inflammation has been
reported to serve a key role in the pathogenesis of VILI (3), the
effects of silencing miR‑127 expression on VILI‑associated
inflammatory responses were investigated. The inflam-
matory response was evaluated via assessing immune cell
numbers and pro‑inflammatory cytokine levels in BAL fluid
samples from mice following mechanical ventilation. The
results demonstrated that, following miR‑127 knockdown,
total inflammatory cell counts were significantly reduced
compared with the VILI+Ad‑shNeg group (P<0.05; Fig. 4A).
Similarly, the levels of the pro‑inflammatory cytokines
TNF‑α, IL‑1β and IL‑6 (P<0.05; Fig. 4B), as well as neutro-
phil counts (P<0.05; Fig. 4C) were significantly decreased
in Ad‑shmiR‑127‑infected mice, compared with the
Ad‑shNeg‑infected mice, following mechanical ventilation.
Silencing miR‑127 expression prevents ventilation‑induced
MPO activation. Mechanical ventilation has been reported
to enhance pulmonary MPO activity, which reflects the accu-
mulation of polymorphonuclear neutrophils in the lungs (23).
The results revealed that following miR‑127 expression
knockdown, MPO activity was significantly suppressed
following mechanical ventilation compared with non‑infected
ventilated mice (VILI group) and mice in the Ad‑shNeg group
(P<0.05; Fig. 4D).
Silencing miR‑127 expression alters the protein expression
of intracellular signaling kinases in VILI. Since miR‑127
knockdown was revealed to suppress the ventilation‑induced
inflammatory response, the molecular mechanisms underlying
its effects were investigated. VILI has been reported to acti-
vate the pro‑inflammatory transcription factor NF‑κ B (14),
and this activation depends on the phosphorylation and subse-
quent degradation of the inhibitor of NF‑κ B, Iκ B (24). NF‑κ B
activation may thus be monitored through the assessment of
Iκ B and p‑Iκ B levels (25,26). In the present study, mechanical
ventilation was demonstrated to increase p‑Iκ B and decrease
Iκ B pulmonary levels (Fig. 4E). Notably, following miR‑127
knockdown, pulmonary levels of p‑Iκ B were downregulated,
4124 LI et al: miR-127 CONTRIBUTES TO LUNG INJURY

Figure 4. Silencing of miR‑127 expression attenuates ventilation‑induced pulmonary inflammation. Mice were infected with either Ad‑shmiR‑127or Ad‑shNeg
adenovirus for 3 days and then mechanically‑ventilated for 6 h. Control mice were allowed to breathe spontaneously and received no ventilation. (A) Total
inflammatory cell counts in BAL fluid samples. (B) Pro‑inflammatory cytokine levels in BAL fluid samples were assessed using ELISA. (C) Neutrophil
counts in BAL fluid samples. (D) MPO activity was assessed as an index of pulmonary neutrophil infiltration in lung tissue samples. (E) Western blot analysis
was used to assess the protein expression levels of Iκ B, p‑Iκ B, p‑p38 MAPK and p‑ERK in lung tissue samples. β‑actin was used as a loading control. Data
are expressed as the mean ± standard deviation (n=18/group). *P<0.05, with comparisons indicated by lines. sh, short hairpin; BAL, bronchoalveolar lavage;
MPO, myeloperoxidase; Iκ B, inhibitor of κ B; p‑, phosphorylated; MAPK, mitogen‑activated protein kinase; ERK, extracellular signal‑regulated kinase; VILI,
ventilator‑induced lung injury; miR, microRNA; TNF, tumor necrosis factor; IL, interleukin.

whereas levels of I κ B were upregulated in ventilated
mice (Fig. 4E), suggesting that NF‑κ B activation may be
suppressed when miR‑127 expression is silenced. In addition,
mechanical ventilation enhanced p38 MAPK phosphorylation
in lung tissue samples, and following miR‑127 knockdown,
p‑p38 levels appeared to be downregulated (Fig. 4E). ERK
phosphorylation was also potentiated as a result of mechanical
ventilation, however, miR‑127 knockdown had no effect on
p‑ERK pulmonary levels (Fig. 4E). These findings suggested
that ventilation‑induced miR‑127 upregulation may contribute
to the pathophysiology of VILI through the activation of the
NF‑κ B‑ and p38 MAPK signaling pathways.
Discussion
The results of the present study suggested that miR‑127 may
contribute to the development of VILI. Mice that received
mechanical ventilation exhibited significantly upregulated
 MOLECULAR MEDICINE REPORTS 16: 4119-4126, 2017
miR‑127 expression in BAL fluid, peripheral blood and lung
tissue samples. Following knockdown of miR‑127 expres-
sion, ventilation‑induced lung damage was significantly
attenuated, and the lung wet/dry ratio and pulmonary inflam-
matory cell infiltration were also decreased. In addition, the
levels of total protein, pro‑inflammatory cytokines and the
pro‑inflammatory factors NF‑κ B and p38 MAPK in BAL
fluid samples, indicative of the presence of VILI, were also
downregulated following the silencing of miR‑127 expres-
sion.
In the present study, miR‑127 expression in mice was
silenced prior to the induction of mechanical ventilation,
through the in vivo delivery of recombinant adenovirus
encoding a miR‑127‑targeting shRNA, as previously
described (27‑29). Infection and gene silencing was successful,
as indicated by the significant decrease in miR‑127 levels in
BAL fluid, peripheral blood and lung tissue samples compared
with mice infected with negative‑control shRNA, prior to and
following mechanical ventilation.
The present results are in accordance with previous studies
that have identified miRNAs as key regulators of numerous
biological processes, including the activation of the innate
immune response (30‑33). Ying et al (14) demonstrated that
lipopolysaccharide‑induced stimulation of RAW 264.7 murine
macrophages in vitro upregulated the expression of miR‑127,
which was suggested to function as a molecular switch during
macrophage development. In the present study, miR‑127
knockdown was revealed to effectively attenuate VILI, which
is characterized by pulmonary edema, neutrophilic inflamma-
tory responses and the release of inflammatory mediators (15).
The present results demonstrated that following miR‑127
knockdown, pulmonary edema was attenuated and the lung wet/
dry ratio, indicative of lung water contents, returned to physi-
ological levels. In addition, MPO activity was suppressed, thus
suggesting that reduced neutrophil sequestration occurred in
the lung, since MPO is a major constituent of neutrophil cyto-
plasmic granules (23). Downregulation of miR‑127 expression
was also revealed to attenuate the ventilation‑induced impair-
ment in alveolar‑capillary barrier permeability, and reduce the
total protein levels in the BAL fluid.
Pro‑inflammatory cytokines which appear in the early
stages of the inflammatory response have been reported
to serve a critical role in the pathogenesis of VILI (34).
Elevated TNF‑ α levels have been reported in BAL fluid
samples isolated from patients with VILI or acute respira-
tory distress syndrome (35,36). IL‑1β has also been identified
as a critical component of VILI pathophysiology (37), as it
has been reported to inhibit fluid transport across the distal
lung epithelium, thus inducing surfactant abnormalities and
increasing the protein permeability of the alveolar‑capillary
barrier. IL‑6 has also been suggested as a marker of VILI (38).
TNF‑ α, IL‑1β and IL‑6 have been reported to initiate and
amplify the inflammatory cascade directly causing inflam-
matory injury, whereas they can also recruit neutrophils into
the lung leading to increased pulmonary MPO activity (3). In
the present study, following knockdown of miR‑127 expres-
sion, ventilation‑induced increases in TNF‑α, IL‑1β and IL‑6
levels were significantly attenuated. These findings suggested
that miR‑127 may contribute to the pathogenesis of VILI, via
stimulating the production of inflammatory cytokines.
4125
NF‑κ B has been identified as a critical regulator of
pro‑inflammatory cytokine expression; activation of the
NF‑κ B, p38 MAPK and ERK signaling pathways has been
suggested to initiate lung inflammatory responses, triggered
by excessive mechanical stress during ventilation (15,39,40).
Under physiological conditions, NF‑κ B is present in the
cytoplasm in an inactive form bound to Iκ B. In response to
mechanical stress in the lungs, Iκ B phosphorylation and subse-
quent degradation is initiated, leading to the release of NF‑κ B,
which then translocates to the nucleus and activates expres-
sion of pro‑inflammatory genes, including TNF‑α, IL‑1β and
IL‑6 (3). Therefore, the present study investigated the effects
of miR‑127 expression manipulations on the activation of
NF‑κ B, p38 MAPK and ERK pathways, which are implicated
in the regulation of cytokine production. The present results
demonstrated that silencing miR‑127 expression appeared
to inhibit the ventilation‑induced activation of NF‑κ B and
p38 MAPK; however, activation of ERK was not affected.
These findings suggested that miR‑127 may contribute to the
development of VILI through the activation of NF‑κ B and p38
MAPK signaling, thus triggering the expression of inflamma-
tory mediators in the lungs.
While the present study demonstrated that miR‑127
positively regulated the inflammatory response during
mechanical ventilation, previous studies involving different
lung injury models have suggested that miR‑127 negatively
regulated inflammatory responses during the development
of lung injury (12,41). The different lung injury models
used in these studies may underlie the apparent discrep-
ancy. In addition, the assessment of miR‑127 expression at
different time points following the induction of lung injury
may have affected the results. Further studies are required
to investigate whether miR‑127 may differentially regulate
inflammatory pathways in different models, or whether it
may exert varying effects at different time points during the
inflammatory process.
In conclusion, the present study suggested that miR‑127 may
contribute to mechanical ventilation‑induced inflammatory
responses in the lungs, through the activation of NF‑κ B and
p38 MAPK signaling pathways, leading to alveolar‑capillary
barrier dysregulation during the pathogenesis of VILI. The
present findings suggested that miR‑127 may have potential
as a novel therapeutic target for the development of strategies
to attenuate VILI in patients receiving mechanical ventilation.
Acknowledgements
The present study was funded by the National Natural Science
Fund (grant no. 81401626).
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